Intermediates Stabilized by Tris(triazolylmethyl)amines in the CuAAC Reaction

Article abstract of DOI:10.1002/chem.201700555

Tris(triazolylmethyl)amine ligands (TL) are widely used to accelerate the Cu^I-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, but its mechanistic role remains unclear. Using electrospray ionization mass spectrometry, we detected for the

Full text of DOI:10.1002/chem.201700555

A Journal of
Accepted Article
Title: Intermediates Stabilized by Tris(triazolylmethyl)amines in CuAAC
Reaction
Authors: Haoqing Chen, Chengzhi Cai, Siheng Li, Yong Ma, Sijin
Luozhong, and Zhiling Zhu
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Intermediates Stabilized by Tris(triazolylmethyl)amines in CuAAC
Reaction
Haoqing Chen, [a] Chengzhi Cai,*[a] Siheng Li,[a] Yong Ma,[b] Sijin Luozhong[c] and Zhiling Zhu[a]
Abstract: Tris(triazolylmethyl)amine ligands (TL) are widely used to
accelerate the CuI-catalyzed azide-alkyne cycloaddition (CuAAC)
reaction, but its mechanistic role remains unclear. Using electro-
spray ionization mass spectrometry, for the first time we detected the
trinuclear TL-CuI3-acetylide and the dinuclear TL-CuI2-acetylide
complexes in aqueous solution. The apparent second-order rate
constants of their reaction with an azide were 27 M-1•s-1 and 783 M-
1•s-1 when the alkyne was tethered to TL. In the catalytic system
without the tether, the rate constant increased to >146 M-1•s-1 for the
TL-CuI3-acetylide, but dropped by ~14 times to ~55 M-1•s-1 for the
TL-CuI2-acetylide. The results indicated that TL accelerated the
reaction by stabilizing the CuI2- and CuI3-acetylide and their azide
adduct intermediates, but this role is largely weakened by excess
alkyne and other competing ligands under catalytic conditions.
comparison, it is more challenging to study the mechanism of
CuAAC reaction accelerated by weaker ligands, such as the
tris(triazolylmethyl)amines[10] (TL, Scheme 1A) that are among
the most efficient and widely used ligands in CuAAC reaction.[2c,
2e, 11] Although a catalytic cycle involving di-CuI acetylides formed
from CuI2TL was proposed (Scheme 1A),[12] the proposed
intermediates were not detected. To facilitate the interception of
the weakly coordinated intermediates, we tethered the TL with
an alkyne via an EG11 chain as in 1 (Scheme 1B). This model
system entropically stabilized the TL-CuI-acetylide intermediates,
allowing us to evaluate their reactivity using ESI-MS.
Introduction
The high efficiency, orthogonality and versatility of the CuI-
catalyzed azide-alkyne cycloaddition (CuAAC, a click reaction)[1]
has rendered it a powerful tool in many fields.[2] The reaction
was proposed to proceed through a rate-limiting transition state
containing two CuI.[3] However, identification of the CuI2-acetylide
and -triazolide intermediates was hampered by the facile ligand
exchange and multiple fast equilibria between the CuI
complexes that are also air-sensitive and prone to aggregation
and disproportionation.[4] To overcome these hurdles, strong CuI
ligands, such as N-heterocyclic carbenes (NHC),[5] cyclic (alkyl)
(amino) carbenes (CAAC),[6] and organophosphines[7] were
employed to stabilize the CuI intermediates, allowing for isolation
of single crystals of NHC-CuI-acetylide cluster,[8] (NHC-CuI)1-
triazolide,[5a] (CAAC-CuI)2-acetylide and (CAAC-CuI)2-triazolide.[6]
Also, organophosphine-stabilized CuI2-acetylide and -triazolide
intermediates were detected by electrospray ionization mass
spectrometry (ESI-MS),[7] an important tool for studying the real-
time distribution of reaction intermediates.[9] Using isotopically
labeled CuI, Worrell et al. demonstrated the necessity of a
second CuI atom to activate the NHC-CuI-acetylide for C-N bond
formation with an organic azide.[5b]
Scheme 1. (A) Previously proposed di-CuI intermediates involved in TL-
accelerated CuAAC reaction,[12] and (B) our model system to facilitate
interception of the intermediates. Charges are omitted.
Results and Discussion
We first attempted to prepare the CuI2TL complex by mixing TL
with [Cu(MeCN)4PF6] (2 equiv.) in MeCN under N2 followed by
lyophilization to completely remove MeCN. After addition of
water, the Cu/TL 2:1 mixture turned into a greenish-yellow
suspension with red precipitates, indicating disproportionation of
the free CuI into CuII and Cu0 (Figure S1A). In comparison, the
aqueous solutions of CuI/1 1:1–3:1 remained colorless and clear
for 4 months (Figure S1B). The ESI-MS spectra of CuI/1 1:1–3:1
solutions (Figure 1A) showed a mixture of CuI-alkyne and -
acetylide complexes. Each major signal was assigned to a CuI
complex ion based on the matches with the predicted m/z value
and isotopic pattern (Figure S3), and their relative abundance
was estimated by the ESI-MS intensities based on the
evidences that indicating these CuI complex ions had a similar
ESI-MS response factor (Figure S5 & Figure 1A). According to
the following discussion, their structures were proposed in
Scheme 2.
The above progress was made using strong CuI ligands. In
[a]
Prof. C. Cai, H. Chen, Dr. S. Li, Z. Zhu,
Department of Chemistry
University of Houston
3585 Cullen Blvd., Houston, Texas 77204-5003, United States
E-mail: cai@uh.edu
[b]
[c]
Dr. Y. Ma
Department of Pharmacological and Pharmaceutical Sciences
University of Houston
1441 Moursund St., Houston, Texas 77030, United States
S. Luozhong
Department of Chemical Engineering
Tsinghua University
Beijing, 100084, China
In the Cu/1 1:1 solution (Figure 1A), the predominant species
was the protonated CuI-alkyne complexes (Ia/Ib). Increasing the
Cu/1 ratio to 2:1 led to a mixture of mostly Ia/Ib, the CuI2-alkyne
complex IIa, and the CuI3-acetylide complex IIIa ([1–H+3Cu]2+).
Supporting information for this article is given via a link at the end of
the document.
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Scheme 2. Proposed mechanism for the reaction of 1 and an azide in the
presence of 1–3 equiv. of CuI. Labels for the observed ions in Figures 1 & 2
are placed under the proposed mono- (I, cyan), di- (II, blue) and tri- (III, red)
CuI complexes. The proposed intermediates in a bracket were not detectable
in the ESI MS. The grey curves represent the long, flexible EG11 linker.
Figure 1. (A) ESI-MS spectra for solutions of 1 (100 μM) and 1–3 equiv. of CuI
generated from CuSO4 (100–300 μM) and Na ascorbate (1 mM) in
deoxygenated water. (B) Averaged ESI-MS spectra of the above solutions
(without NaOH) taken between 4–6 min after addition of 1 equiv. of N3EG4. All
marked peaks are assigned and color-coded with cyan for mono-CuI, blue for
di-CuI and red for tri-CuI species (Scheme 2). The assignment is based on a
good fit of the experimental (solid lines) and theoretical (dash line) isotopic
patterns (Figures S3 & S4, selected examples are shown in inserts).
Only ~5% of the deprotonated CuI2-acetylide IIb was present. At
the ratio of Cu/1 3:1, IIIa became the predominant species (see
also Table 1). Addition of NaOH produced more CuI2-acetylide
IIb (Figures 1A, S7 and Table 1). Overall, the stability of
acetylides in aqueous solution increased as the number of CuI
coordination became higher (stability: IIIa > IIb > Ic). Indeed, IIIa
was present even in the Cu/1 1:1 mixture at pH 6, while Ic was
not detectable (see Figure S8 for the amplified MS spectra).
The coordination mode of IIIa was proposed (Scheme 2)
Figure 2. (A) Time course of ESI-MS intensities of the assigned intermediates
during the reaction of 1 with N3EG4. Conditions: 100 μM 1, 300 μM CuSO4,
100 μM N3EG4 and 1 mM Na-Ascorbate in deoxygenated water at room
temperature. For the labels, see Scheme 2. (B) Apparent second order rate
constants (kobs) for reaction of 1 with N3EG4 quantitatively measured by LC-MS.
Conditions: 100 μM 1, 100 μM N3EG4 and
1 mM Na-ascorbate in
deoxygenated water at room temperature, with: 100 μM CuSO4 (cyan); 200
μM CuSO4 (blue); 300 μM CuSO4 (red); 600 μM CuSO4 & 500 μM NaOH
(green); 200 μM CuSO4 & 500 μM NaOH (purple); and 100 μM CuSO4 & 500
μM NaOH (navy). Data from Figure S15.
based on the crystal structure of
a
TL-CuI3-acetylide
analogue.[13] The interaction between CuI and alkyne or acetylide
in the Cu/1 mixtures in D2O was evidenced by the large 1H NMR
downfield shift (0.19–0.58 ppm) of the propargylic protons
(Figure S6).
azide-CuI2-acetylide adduct was reported,[7] the unstable adducts
IIc1 and IIIb1 in our system (Scheme 2) were not detectable.
The reactivity of the unprecedented tri-nuclear complex IIIa
was first investigated by monitoring the reaction of the Cu/1 3:1
mixture (containing ~86% IIIa, Table 1) with 1 equiv. of N3EG4.
As shown in Figure 2A, the MS intensity of IIIa dropped
Addition of 1 equiv. of the azide N3EG4 (Scheme 1B) to the
solutions of Cu/1 1:1–3:1 generated new signals (Figure 1B)
assigned to the Cu2-triazolide IIc and Cu3-triazolide IIIb, and the
Cu-triazole complexes 2-Cu2 and 2-Cu (Scheme 2, see Figures
S4 & S9–S11 for the isotopic patterns and tandem mass
spectrometry analysis). Although an organophosphine-stabilized
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Table 1. MS Intensities of the CuI-acetylides in Ligand
Observed Rate Constants (kobs) vs. Calculated Rate Constants (kcal
1 System, and
drastically during the first 10 min, accompanied by a rapid
increase of the CuI3-triazolide IIIb whose signal intensity
exceeded that of IIIa after 10 min, then decreased after ~15 min.
Consumption of IIIa and accumulation of IIIb as the most
abundant intermediate were faster than the formation of the
triazole complexes 2-Cu2 and 2-Cu, which was possibly via
hydrolysis of IIIb to the transient 2-Cu3 followed by dissociation
of CuI from 2-Cu3 to 2-Cu2, or from IIIb to IIc followed by
hydrolysis (Scheme 2).
[a]
)
ESI MS Intensity / 107
[a]
Cu/1/
kobs
kcal
NaOH[b]
M-1S-1
M-1S-1
Ia/Ib
149.0
45.1
4.3
IIb
1.5
IIIa
0.8
IIa
5.3
31.6
12.3
0
1:1:0
2:1:0
3:1:0
1:1:5
2:1:5
6:1:5
9
8
5.4
43.7
132.0
0
41
43
4.7
47
47
The coordination mode of Cu3-triazolide IIIb is tentatively
proposed in Scheme 2 and Figure S14. The three coordinating
CuI ions greatly stabilized IIIb against hydrolysis compared to
the Cu2-triazolide IIc (showing a low intensity throughout the
reaction, Figure S13) and the Cu-triazolide Ic (not detectable).
We next compared the reactivity of the tri-copper acetylide IIIa
with the di-copper acetylide IIb as the key intermediate in the
proposed mechanism.[3] To enrich the deprotonated IIb, 5 equiv.
NaOH was added to the solution of Cu/1 2:1 generating the
highest percentage of IIb (67%, Table 1) among all Cun-
alkyne/acetylide species (Figure S7); further addition of the base
promoted formation of dimers and CuOH. The stoichiometric
reactions of 1 with N3EG4 in the presence of 1–6 equiv. of CuI
(and 5 equiv. of NaOH) were quantitatively analyzed by LC-MS
and their apparent second order rate constants (kobs) were
determined (Figures 2B, S15, and Table 1). It is generally
agreed that the first C-N bond formation from the Cu2-acetylide-
azide adduct is the rate-limiting step for CuAAC reaction.[3d-f, 14]
We assume that the C-N bond can be formed through the di-
copper and the tri-copper pathways via the azide adducts IIc1/
IIIb1 (Scheme 2), and their barrier is much higher than those of
the pre-equilibria. Under these assumptions, Eq. (1) derived
from a generalized pre-equilibrium approximation method[15] and
the data in Table 1 can be used to extract the apparent rate
constants for converting IIb to IIc (k’II) and IIIa to IIIb (k’III):
47.2
40.2
1.2
45.9
67.2
6.3
386
471
38
386
483
50
0.8
0.7
5.0
185.0
[a] Rate constant calculated using Eq. (1), in which k’II = 783 M-1s-1 and k’III
27 M-1s-1. [b] The molar ratio of added NaOH.
=
measured with this system better represents the barriers for the
C-N bond formation than with a catalytic system (see below).
Overall, this model system showed a much higher kobs (9–471
M-1•s-1) than the previously reported values (kobs = 0.002 3.5 M-
1•s-1) for CuAAC reaction accelerated by TL ligands (without the
linked alkyne, see Scheme 1A for structure of TL).[12b] Compared
to the above model system, reaction of the alkyne 3 and the
azide N3EG4 under catalytic conditions (Figure 3A) showed
much lower kobs values of 2.1–3.6 M-1•s-1 at 3/Cu = 10:6 (Table
S2), which further decreased to 0.3–0.4 M-1•s-1 at lower catalyst
loading (3/Cu = 100:6, Table S3).
First of all, both the di-Cu acetylide (iia, [3 H+TL+2Cu]+) and
tri-Cu acetylide (iiia, [3 H+TL+3Cu]2+) were present under
catalytic conditions (Figures 3B, S17 and S22). The ratio of
[iiia]/[iia] increased with higher Cu/TL ratio and catalyst loading
(Tables S2 and S3). Without TL, no di- and tri-Cu acetylide was
detected (Figure S17) and the reaction was 25 times slower
(Figure S20), consistent with the role of TL to stabilize these
active species.
kobs = xIIbk’II + xIIIak’III
Eq. (1)
in which xIIb and xIIIa are the molar fraction of IIb and IIIa derived
from the ESI-MS intensities of the intermediates. Fitting the data
to Eq (1) gives k’II = 783 M-1•s-1 and k’III = 27 M-1•s-1 (Table S1)
with a good agreement between the calculated rate constants
kcal and the measured kobs (Table 1). The ratio of k’II/k’III = 29:1
agreed with result of our DFT calculation showing that the
barrier for C-N bond formation via a Cu2 transition state was ~ 2
kcal/mol lower than via a Cu3 transition state (unpubilished
result).
The apparent rate constants k’II and k’III depend primarily on
the energy barriers for the C-N bond formation, but they can also
be significantly affected by any hidden equilibria interfering the
transformation. These equilibria include dissociation of the azide
adduct and its association with other ligands in the system,
which are minimized in the model system. Specifically, the ~50 Å
EG11 linker in 1 maintained a relatively high local concentration
(in millimolar range) of the alkyne, which stabilized the TL-Cun-
acetylides (IIIa/IIb) and their azide adducts (IIIb1/IIc1) against
dissociation, and yet should not change the barrier for the C-N
bond formation. In the Cu/1 ≥ 2 system, there was no extra
ligand interacting with the di- and tri-Cu acetylides and their
azide adducts. Hence, the apparent rate constants k’II and k’III
Figure 3. (A) CuAAC reaction of the alkyne 3 and RN3 (R = EG4). (B) ESI-MS
spectra for the solutions of 3 (100 μM), TL (20, 30, 60 μM), CuI (60 μM) and
Na-ascorbate (0.5 mM) in deoxygenated water. For assignment of the labeled
ions, see Figure S18 & S19. (C) Effect of CuI/TL ratio on reaction order to [Cu],
with 3 (100 μM), N3EG4 (100 μM), CuI (30, 60, 90, 120, 180, 240, 300 μM),
Cu/TL (1:1, 2:1, 3:1). See also Table S2 for the amount of iiia and iia vs kobs at
3/Cu = 10:6 (boxed in C, data taken from B).
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In summary, we reported the first example of tri-Cu-acetylide
as a key intermediate in CuAAC reaction. A similar intermediate
was proposed by Meldal and Tornøe.[2a] Compared to the di-Cu-
acetylides, the tri-Cu-acetylide showed superior stability against
protonation in aqueous solution at neutral pH. The TL ligand
stabilizes the TL-CuIn-acetylide (n = 2, 3) complexes and their
azide adducts as the reactive intermediates, but the strength of
stabilization is limited, especially at a low catalyst loading where
the complexes are destabilized by the large excess of alkyne
and other competing ligands.
Experimental Section
ESI-MS analysis for intermediate solution equilibria in CuIn1
solutions w/ or w/o N3EG4 (Figure 1 & Figure 2A)
Scheme 3. Proposed mechanism for the CuAAC reaction of 3 and RN3 (R =
EG4) in the presence of catalytic amount of CuI/TL. For the intermediates
detected by ESI-MS, see Figures 3, S17-S19 and S22; intermediates in a
bracket were not detectable. L = ligands 3 and TL, etc.
ESI-MS spectra of CuIn1 solutions and the CuAAC reaction
mixtures (with N3EG4) were acquired using a Thermo Finnigan
LCQ Deca XP Plus ion trap mass spectrometer at capillary
temperature of 40°C, spray voltage 4 kV, capillary voltage 28 V,
multipole 1 offset 3 V, multipole 2 offset 12 V, entrance lens
voltage 61 V. In an anaerobic chamber, all stock solutions were
prepared in deoxygenated Milli-Q water.
The slow kinetics under catalytic conditions can be attributed
mainly to the dissociation of the Cun-acetylides iia/iiia and their
azide adducts iib/iiib in which the Cu-acetylide bond was
weakened by azide-coordination.[14] The dissociation was
enhanced both by the increased entropy (compared to the EG11-
tethered 1 system) and by the excess alkyne and TL (Scheme
3), generating inactive Cu complexes 3-Cu, TL-Cu, and Cu-
acetylide aggregates (Figures 3 and S17). These equilibria led
to fractional rate orders in [Cu] (especially at a low Cu/TL ratio,
Figure 3C), and greatly decreased the apparent rate constant
for the C-N bond formation via the di-Cu pathway (k’II) by ~14
times to ~55 M-1•s-1 (Tables S2 and S3).
In sharp contrast to the di-Cu pathway, the apparent rate
constant for the C-N bond formation via the tri-Cu pathway (k’III)
was increased by 5 9 times to 146 260 M-1•s-1 (Tables S2 and
S3). Accordingly, the k’II/k’III ratio drastically dropped from 29 in
the model system to <0.4 in the catalytic system (Tables S1-S3),
showing a greatly enhanced contribution to kobs by the tri-Cu
acetylide iiib (for an example, see Figure 3B,C). To rationalize
this result, we note that compared to the di-Cu acetylide iib, the
tri-Cu acetylide iiib has higher affinity with the negatively
charged internal nitrogen of azide.[3b, 3g, h, 10f, 16] As illustrated in
Scheme 3, ligand coordination to the tri-Cu-acetylide-azide
adduct iiib will facilitate the dissociation of a CuI (internally) from
the acetylide iiic to generate a di-Cu transition state TSCu2 via iib
or TS’Cu2 (through the internal dissociation) for C-N bond
formation. Furthermore, the triazolides iic and iiid (detected by
ESI-MS, Figure S22) may serve as a base to deprotonate the
alkyne 3, especially those formed from TS’Cu2 in which the
coordinated alkyne is proximal to the triazolide, to generate the
product 4 and regenerate the copper acetylides iia and iiia
(Scheme 3).
To study the solution equilibria of CuIn1 complexes (Figure 1A),
100 μL 1 mM ligand 1, 100/200/300 μL 1 mM CuSO4, 100 μL 10
mM Na ascorbate, and 700/600/500 μL deoxygenated water
were mixed in 3 separate Eppendorf tubes in an anaerobic
chamber, vortexed, and then directly infused into the ESI source
by a syringe pump at a flow rate of 10 μL/min. Each full MS
spectrum was an average of 100 individual scans.
In separate experiments, to promote the deprotonation of IIb
(Figures 1A, S7 and Table 1), 100 μL 1 mM ligand 1,
100/200/600 μL 1 mM CuSO4, 100 μL 10 mM Na ascorbate, 100
μL 5 mM NaOH, and 600/500/100 μL deoxygenated water were
mixed in 3 separate Eppendorf tubes and the spectra were
recorded using the above procedure.
To study the solution equilibria of CuAAC reaction (Figure 1B),
100 μL 1 mM ligand 1, 100/200/300 μL 1 mM CuSO4, 100 μL 1
mM azido-tetraethylene glycol (N3EG4), 100 μL 10 mM Na
ascorbate, and 600/500/400 μL deoxygenated water, were
mixed in 3 separate Eppendorf tubes in anaerobic chamber,
vortexed, and then immediately infused into ESI source by a
syringe pump at a flow rate of 10 μL/min. Reaction time was
recorded on a timer. Each full MS spectrum in Figure 1B was an
average of scans during the reaction time of 4–6 min. The
reaction mixture was continuously infused into the ESI source
during a period of 20 min. At Cu/1 = 3:1, the variation of ESI-MS
intensities of selected intermediates during the reaction was
plotted on Figure 2A.
Kinetic studies for reaction between 1 and N3EG4 (Figure
2B)
The reactions were conducted in an anaerobic chamber at room
temperature. Solutions in 900 μL deoxygenated Milli-Q water
was added into separate Eppendorf tubes, containing 100 nmol
1, 1 μmol sodium ascorbate, and: A: 100 nmol CuSO4, B: 200
nmol CuSO4, C: 300 nmol CuSO4, D: 100 nmol CuSO4 and 500
nmol NaOH, E: 200 nmol CuSO4 and 500 nmol NaOH, F: 600
Conclusions
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nmol CuSO4 and 500 nmol NaOH. 100 μL 1 mM azido
tetraethylene glycol (N3EG4) aqueous solution was added into
each tube to initiate the reaction. At 20 s (for A, B, C) or 10 s (for
D, E, F) intervals, 20 μL aliquots of the reaction mixture were
added into a 96-well plate, each well containing 180 μL air-
saturated aqueous solution of 20 nmol diethylene triamine
pentaacetic acid (DTPA) (pH adjusted to 7 by NaOH) as the
quenching reagent and 1 nmol 5 (structure shown in Scheme
S1) as internal standard. After 8 aliquots were collected, the 96-
well plate was moved out of anaerobic chamber. The samples
were injected to a Thermo Finnigan LCQ Deca XP Plus ion trap
mass spectrometer through a Thermo Finnigan Surveyor HPLC
system. Reagents and product were separated by first static
elution in 3% (MeCN, 0.1% formic acid) : 97% (H2O, 0.1% formic
acid) for 5 min and then a linear gradient elution to 95% (MeCN,
0.1% formic acid) : 5% (H2O, 0.1% formic acid) for 5 min. A set
of calibration standards of product 2, ranging from 100 nM to 10
µM, using 5 as internal standard, was injected before the
unknowns. The concentration of product 2 in each sample was
calculated based on the calibration curve. Each reaction under
same condition was repeated three times.
mixture were added into a 96-well plate, each well containing 90
μL air-saturated diethylene triamine pentaacetic acid (DTPA)
aqueous solution (pH adjusted to 7.0 by NaOH). After 6 sets of
aliquots were collected, the 96-well plate was moved out of
anaerobic chamber. 10 µL 100 µM S10 (structure shown in
Scheme S1) water solution was added into each aliquot as
internal standard. The samples were injected to a Thermo
Finnigan LCQ Deca XP Plus ion trap mass spectrometer
through a Thermo Finnigan Surveyor HPLC system. Reagents
and product were separated by first static elution in 3% (MeCN,
0.1% formic acid) : 97% (H2O, 0.1% formic acid) for 2 min and
then a linear gradient elution to 90% (MeCN, 0.1% formic acid) :
10% (H2O, 0.1% formic acid) for 5 min. A set of calibration
standards of product 4, ranging from 500 nM to 50 µM, using
S10 as internal standard, were injected before and after each
set of samples. The concentration of product in each sample
was calculated based on the calibration curve.
Acknowledgements
This work was supported by the National Institute of Health grant
(5R01EY013175), the National Science Foundation grant (DMR-
1508722), and the GEAR grant from the University of Houston.
We thank Dr. R. Thummel, Dr. S. Bark and Dr. M. Hu for
permission and help to use their equipment.
ESI-MS analysis for intermediate solution equilibria in CuIn3
solutions (Figure 3B, S17)
ESI-MS spectra of CuIn3 solutions were acquired using a
Thermo Finnigan LCQ Deca XP Plus ion trap mass
spectrometer at capillary temperature of 40°C, spray voltage 4.5
kV, capillary voltage 32 V, multipole 1 offset 5V, multipole 2
offset 12V, entrance lens voltage 51 V. In an anaerobic
chamber, all stock solutions were prepared in deoxygenated
Milli-Q water.
Keywords: Click Chemistry • CuAAC Reaction • Reactive
Intermediate • Mass Spectrometry • Tri-copper Acetylide
[1]
[2]
a) V. V. Rostovtsev, L. G. Green, V. V. Fokin and K. B. Sharpless,
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Chem. 2002, 67, 3057 3064.
To study the solution equilibria of 3/Cu = 10:6 solution (Figure
3B), 50 μL of 10 mM sodium ascorbate was added into 950 μL
of aqueous solution containing 100 nmol 3, 60 nmol CuSO4, and
60/30/20 nmol TL.
To study the solution equilibria of 3/Cu = 100:6 solution
(Figure S17), 50 μL of 10 mM sodium ascorbate was added into
950 μL of aqueous solution containing 1 μmol 3, 60 nmol CuSO4,
and 120/60/30/20/15/0 nmol TL.
The mixtures were directly infused into ESI source by a
syringe pump at a flow rate of 10 μL/min. Each full MS spectrum
was an average of about 100 individual scans. Zoom scan
analysis was carried out to determine the charge state and
isotopic pattern for selected CuI adducts. Each zoom scan
spectra represents an average of 50–100 individual scans
(Figure S18). MS/MS analysis were carried out to determine the
fragmentation pattern of selected CuI adducts (Figure S19).
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Tri-Cu acetylides and triazolides in
CuAAC: Mechanistic study of the
tris(triazolylmethyl)amine ligand
accelerated CuAAC reaction was
performed in aqueous solutions.
Reactive tri-Cu and di-Cu
Haoqing Chen, Chengzhi Cai*, Siheng
Li, Yong Ma, Sijin Luozhong, Zhiling Zhu
Page No. – Page No.
Intermediates Stabilized by
Tris(triazolylmethyl)amines in CuAAC
Reaction
intermediates were identified by ESI-
MS and their CuAAC activity were
quantitatively measured by LC-MS.
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